The present invention is related to the field of vaccines and is particularly related to the development of novel glycoconjugation technologies which can be used to prepare glycoconjugates in which multi-oligosaccharides are covalently linked to the same carrier protein.
Haemophilus influenzae type b (Hib), Neisseria meningitidis and Streptococcus pneumoniae are major causes of bacterial meningitis in children under five years of age. All these bacteria are protected from phagocytosis by a polysaccharidic capsule. Antibodies induced against the capsular polysaccharide (CPs) of the organism are protective in most cases. Effective Hib conjugate vaccines in which Hib CPs, PRP, is linked to different carrier proteins, such as diphtheria toxoid (PRP-D), tetanus toxoid (PRP-T), CRM 197 (HbOC) and the outer membrane proteins of N. meningitidis (PRP-OMP), have been developed. Four Hib conjugate vaccines are now commercially available. New glycoconjugate vaccines against N. meningitidis and S. pneumoniae are highly recommended by the American College of Physicians.
The development of multivalent pneumococcal vaccines for the prevention of both systemic and noninvasive pneumococcal diseases in infants, the elderly and immune-compromised individuals has gained increasing importance over the last decade. For more detailed reviews of pneumococcal disease, epidemiology, or the polysaccharide vaccine, numerous review articles are available (ref. 1, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure).
Streptococcus pneumoniae is a capsulated, gram-positive bacterium that is present as normal flora in the human upper respiratory tract. It is a Frequent and major cause of pneumonia, meningitis, bacteremia and noninvasive bacterial otitis media. Disease incidence is highest in infants and the elderly. In the United States alone, the overall incidence of systemic pneumococcal infections is estimated to be 50/100,000 in the geriatric population and 160/100,000 in children less than 2 years old (refs. 2, 3). Case fatalities can be as high as 40,000/year, especially in the geriatric population. Many serotypes of S. pneumoniae are developing resistance to conventional antibiotic treatments. The incidence of otitis media in children approaches 90% by the age of 5 and the peak incidence occurs at 6 to 15 months of age. It was estimated that over 1.2 million cases of otitis media occur annually. Recent studies on the epidemiology of pneumococcal disease (ref. 4) have shown that five serotypes (6B, 14, 19F, 23F and 18C) of the 85 known serotypes account for 70 to 80% of pneumococcal disease in infants and that in the United States, types 9V and 4 are ranked sixth and seventh. In Europe and developing countries, types 1 and 5 are more prevalent than types 4 and 9V. Thus, a pneumococcal conjugate vaccine for the United States should contain at least seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) to achieve a 75 to 85% coverage. Conjugate vaccine formulations for Europe and elsewhere should include serotypes 1, 5, 6B, 14, 18C, 19F and 23F. A broad-spectrum multivalent pneumococcal conjugate vaccine should then contain CPs from nine serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F.
N. meningitidis is a gram-negative bacterium that has been serologically classified into groups A, B, C, 29e, W135, X, Y and Z. Additional groups (H, I, and K) were isolated in China and group L was isolated in Canada. The grouping system is based on the capsular polysaccharides of the organism. In contrast to the pneumococcal vaccine, the composition of the meningococcal polysaccharide vaccine has been greatly simplified by the fact that fewer polysaccharides are required. In fact groups A, B, and C are responsible for approximately 90% of cases of meningococcal meningitis. Prevention of group A and C meningococcal meningitis can be achieved by vaccination with a bivalent polysaccharide vaccine. This commercial vaccine has been used successfully in adults during the last decade to prevent major meningitis epidemics in many parts of the world. However, there is a need to improve this vaccine because a significant proportion of cases of meningococcal meningitis are due to serotypes other than A and C. Group B N. meningitidis is of particular epidemiologic importance, but groups Y and W135 are also significant. Although a tetravalent vaccine comprising groups A, C, W135, and Y polysaccharides is the current meningococcal meningitis vaccine, it is not very effective in young infants, since maturation of the immune response to most capsular polysaccharides in infants occurs around the age of 2 years.
The Group B meningococcal polysaccharide is poorly immunogenic in man. Two major reasons have been proposed to account for this phenomenon. One is that the xcex1-(2xe2x86x928)-linked sialic acid homopolymer is rapidly depolymerized in human tissue by neuraminidase. The other one is that Group B capsular polysaccharide is a polymer of N-acetylneuraminic acid (xcex1 2xe2x86x928 NeuNAc), and that the xcex1 2xe2x86x928 NeuNAc moiety is found as a monomer and dimer on several glycoproteins and gangliosides in adults and as a polymer of at least eight repeating units in rat fetal and newborn tissues. Thus, this structure is recognized as a xe2x80x9cselfxe2x80x9d antigen by the human immune system. As a result, the production of antibody is suppressed or because of this molecular mimicry, a vaccine based on native Group B CPs might induce auto-antibodies directed against the xcex1 2-8 NeuNAc moiety, and thus cause autoimmune diseases.
Since the Group B meningococcal CPs is not immunogenic in humans, approaches have been pursued to increase its immunogenicity. One approach uses non covalent complexes of Group B CPs and outer membrane protein (OMPs). Such complexes are formed by hydrophobic interaction between the hydrophobic regions of the OMPs and the diacyl glycerol group at the reducing end of the CPs. Human volunteers were given two doses of the complex at 0 and 5 weeks. Most individuals responded with an increase in antibodies to group B CPs. However, the second dose resulted in little or no increase in antibody titres which subsequently declined over a period of 14 weeks. The antibodies with group B polysaccharide specificity were limited to the IgM class and directed against determinants present only on high molecular weight polysaccharides.
To improve the immunogenicity of Group B CPs, Jennings (ref. 5) prepared a Group B meningococcal-tetanus toxoid conjugate (GBMP-TT) by covalently linking the CPs to tetanus toxoid (TT) through its terminal non-reducing sialic acid using periodate oxidized CPs. This procedure, however, did not result in any significant enhancement in CPs immunogenicity. The antibody response elicited in animals was found to be primarily directed against the linkage point between the CPs and the protein (GBMP-TT). Further improvement of the immunogenicity of group B CPs involved its chemical modification. Jennings (Ref. 6) reported that the N-acetyl groups of group B CPs could be selectively removed by the action of a strong base at elevated temperature. The acetyl groups were then replaced with N-propionyl groups by propionic anhydride treatment to produce N-propionylneuraminic acid (xcex1 (2xe2x86x928) NeuPro) polymers. The N-propionylated CPs was first periodate oxidized with sodium periodate, and then coupled to TT in the presence of sodium cyanoborohydride to yield the chemically modified GBMP-TT conjugate. Mice immunized with this conjugate formulated in Freund""s complete adjuvant (FCA), generated high levels of cross-reactive IgG antibody against native group B CPs. Murine anti-sera were found to be bactericidal for all group B strains. However, further studies revealed the existence of two populations of antibodies with different specificity. One population reacted with purified group B CPs whereas the other one did not. Antibodies that did not react with native group B CPs appeared to be responsible for bactericidal activity. These antibodies may recognize an epitope expressed by cell-associated CPs that is not present on purified CP. Alternative conjugates comprising the capsular polysaccharide of N. meningitidis group B CPs conjugated to a carrier protein as immunogenic compositions, including vaccines, and their use as and for the generation of diagnostic reagents, had been described by Kandil et al. (U.S. Pat. No. 5,780,606, assigned to the Assignee hereof and the disclosure of which is incorporated herein by reference, EP 0747063). In particular, the capsular polysaccharides of N. meningitidis contain multiple sialic derivatives that can be modified and used to attach carrier molecules.
The dramatic reduction in Haemophilus influenzae type b disease observed in countries that have licensed and used Hib CPs-protein conjugate vaccines, has demonstrated that CPs-protein conjugates can prevent systemic bacterial diseases. It is reasonable to expect that meningococcal and pneumococcal CPs-protein conjugates will also be efficacious. The possibility of preventing noninvasive diseases, such as otitis media, by systemic immunization with conjugate vaccines needs to be explored. Whether high titers of serotype-specific antibodies are sufficient to prevent either nasopharyngeal colonization and/or otitis media remains an open question. The development of an otitis media vaccine requires a multiple pneumococcal CPs-protein conjugates to elicit high anti-CPs antibody titers early in life.
The development of both multivalent pneumococcal and meningococcal CPs-protein conjugate vaccines to prevent systemic and noninvasive diseases presents many challenges to carbohydrate chemists, immunologists, clinicians and vaccine manufacturers. The amount of carbohydrate, the choice of carrier, the method of vaccine delivery, and the use of immuno stimulants or adjuvants are known to influence on the host immune responses. Immunogenic glycoconjugates can be formed between multifunctionalized CPs and proteins if the conditions are controlled very carefully. Most of the conjugates are today synthesized by coupling either CPs or oligosaccharides activated through the reducing end to a protein or peptide with or without a linker group.
A general glycoconjugation method involves random activation of the capsular polysaccharide or fragments of the polysaccharide by periodate treatment. The reaction leads to a random oxidative cleavage of vicinal hydroxyl groups of the carbohydrates with the formation of reactive aldehyde groups. Coupling to a protein carrier is by direct amination to the lysyl groups. A spacer group, such as aminocaproic acid, can be reacted with the aldehydes by reductive amination and then coupled to the protein lysyl groups by water soluble carbodiimide condensation (ref. 7). The oligosaccharide-peptide conjugate reported by Paradiso (ref. 8) was prepared similarly except that a peptide presenting a T-cell epitope of CRM197 was used instead of the native protein. Other conjugation approaches that have been disclosed by Gordon in U.S. Pat. No. 4,496,538, assigned to Connaught Laboratories Inc., and by Schneerson et al. (ref. 9), involve directly derivatizing the CPs with adipic acid dihydrazide (ADH) following CNBr activation, and then conjugating the derivatized CPs directly to a carrier protein (D or T) by carbodiimide condensation. Marburg and Tolman (EP#534764A1) demonstrated that protein-dimeric CPs conjugate immunogens may be produced by coupling the first CPs to a protein carrier and then linking the second CPs to the first CPs via a bifunctional cross-linker.
Methods for inducing immunity against disease are constantly improving. Research has focused on the structure-function relationship of carbohydrate protein conjugates with the hope of discovering the mechanisms of B- and T-cell interactions with conjugates that could lead to vaccines with improved immunogenicity and to the development of adjuvants and delivery systems. Chong et al. (U.S. Pat. No. 5,679,352, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference) showed that several factors affect the immunogenicity of carbohydrates. The minimum requirements for the synthesis of an immunogenic glycoprotein conjugate are that the B-cell epitope(s) of the CPs and the T-cell epitope(s) of the carrier should be functional after covalent linkage. The magnitude of the anti-CPs antibody response markedly depends on the spatial orientation of CPs relative to the T-cell epitopes. Anti-CPs antibody responses are enhanced when multiple antigenic peptides (MAPs) are used as carriers.
A single-dose polyvalent vaccine is listed as the first priority in the WHO vaccine development programme. A single-dose polyvalent CPs-protein conjugate vaccine (15 different CPs-protein conjugates: 1 Hib conjugate, five N. meningitidis conjugates and nine S. pneumoniae conjugates) against bacterial meningitis, presents a potential risk of hyperimmunization against classical carrier proteins, such as diptheria and tetanus toxoids. It is documented that non-epitope-specific suppression of the antibody response to Hib conjugate vaccines resulted from pre-immunization with carrier proteins (ref. 11). Thus, appropriate approaches are required to solve this vaccine formulation problem. Some of the problems can be circumvented by incorporating conserved, cross-protective, non-capsular antigens from Hib, N. meningitidis and S. pneumoniae. Although several outer membrane proteins have been proposed as vaccine candidates, none of them has been tested in clinical trials. A multiple CPs-carrier conjugate delivery system thus represents a novel generic approach and will be important in glycoconjugate vaccine development. Therefore, the present invention is directed towards novel glyconjugation technologies which can be used to prepare vaccines containing multiple oligosaccharides from different bacteria covalently linked to the same carrier protein or polypeptide.
In accordance with one aspect of the present invention, there is provided a multivalent immunogenic molecule, comprising a carrier molecule containing at least one functional T-cell epitope, and multiple different carbohydrate fragments each linked to the carrier molecule and each containing. at least one functional B-cell epitope, wherein said carrier molecule imparts enhanced immunogenicity to said multiple carbohydrate fragments.
In one embodiment of the invention, the carbohydrate fragments are bacterial capsular oligosaccharide fragments. Such capsular polysaccharide fragments may be oligosaccharide fragments of Streptococcus pneumoniae, including fragments derived from at least two capsular polysaccharides of S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. The carrier molecule may be a T-cell epitope-containing protein or protein fragment of S. pneumoniae. 
The capsular polysaccharide fragments may be oligosaccharide fragments of Neisseria meningitidis, including fragments derived from at least two capsular polysaccharides of N. meningitidis Groups A, B, C, W-135 and Y. The carrier molecule may be a T-cell epitope-containing protein or protein fragment of N. meningitidis. 
The capsular polysaccharides employed in this aspect of the invention may be oligosaccharide fragments sized from about 1 to about 5 kDa. Such fragments may be provided by acid hydrolysis of the respective capsular polysaccharide. The oligosaccharide fragments may be chemically modified for coupling to the carrier molecule.
The carrier molecule may be an oligopeptide containing at least one functional T-cell epitope or a carrier protein, such as tetanus toxoid.
In another embodiment of the invention, the carbohydrate fragments are fragments of carbohydrate-based tumor antigens. Such carbohydrate-based tumor antigens may be Globo H, LeY or STn.
In accordance with another aspect of the invention, there is provided a method of forming a multivalent immunogenic molecule, comprising treating at least two different carbohydrate molecules to obtain carbohydrate fragments thereof, and conjugating each of the carbohydrate fragments to a carrier molecule.
In one embodiment, the carbohydrate molecule is a capsular polysaccharide of a bacteria and oligosaccharide fragments of the capsular polysaccharide are selected sized from 2 to 5 kDa. Such oligosaccharide fragments generally are derived from at least two different serotypes of the same bacteria, including S. pneumoniae and N. meningitidis. 
In this embodiment of the present invention, such multivalent immunogenic molecules may be provided by glycoconjugation of three or more chemically-activated capsular polysaccharides or their derivations simultaneously to a single carrier molecule, providing a random conjugation. This procedure is illustrated in FIG. 1.
In this embodiment of the invention, rational design of lysine-branched peptide systems may be employed for site-directed glycoconjugation. Using different side-chain protecting groups for lysine and cysteine residues during peptide synthesis, activated oligosaccharides may be selectively and sequentially linked to the same carrier molecule through such residues. This procedure is illustrated in FIG. 2.
The method of site-directed conjugation may comprise first forming a multiple antigen peptide as the carrier molecule and anchored to a polymeric anchor wherein at least two carrier peptide segments have different terminal protecting groups. One of the protecting groups then is selectively removed and a first one of the oligosaccharide fragments is coupled to the unprotected carrier peptide segment. Another of the protecting groups is selectively removed and a second one of the oligosaccharide fragments to the unprotected carrier peptide segment. This procedure may be repeated for as many carrier peptides and oligosaccharide fragments as is provided and in respect of which coupling is desired. The resulting molecule is cleaved from the polymeric anchor.
In accordance with a further aspect of the invention, there is provided an immunogenic composition for protection against meningitis, comprising (1) a multiple pneumccoccal glycoconjugate according to claim 3, (2) a multiple meningococcal glycoconjugate according to claim 6, and (3) an immunogenic synthetic PRP-peptide conjugate.
The multiple pneumococcal glycoconjugate may be derived from at least two capsular polysaccharides of S. pneumoniae serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F. The multiple meningococcal glycoconjugate may be derived from at least two capsular polysaccharides of N. meningitidis Groups A, B, C, W-135 and Y.
Such universal meningitis immunogenic composition may be combined with at least one other antigen, such as DTP-polio, to provide a polyvalent vaccine.
The present invention further includes a method of generating an immune response in a host by administering to the host an immunoeffective amount of an immunogenic composition of the present invention. The invention extends to the immunogenic composition claimed herein when used as a medicament against meningitidis as well as the use of the individual component of the immunogenic composition in the manufacture of a medicament against meningitidis.
The present invention further includes diagnostic procedures and kits using the multivalent immunogenic molecules provided herein. Accordingly, in an additional aspect of the invention, there is provided a method of determining the presence of antibodies specifically reactive with a multivalent immunogenic molecule as provided herein, which comprises:
(a) contacting the sample with said multivalent immunogenic molecule to produce complexes comprising the molecule and any said antibodies present in the sample specifically reactive therewith; and
(b) determining production of the complexes.
In a further aspect of the invention, there is provided A diagnostic kit for determining the presence of a multivalent immunogenic molecule as provided herein, comprising:
(a) the multivalent immunogenic molecule;
(b) means for contacting the multivalent molecule with the sample to produce complexes comprising the multivalent molecule and any said antibodies present in the sample; and
(c) means for determining production of the complexes.
The present invention, therefore, permits pneumococcal glycopeptide conjugates to be used in a diagnostic immunoassay procedure or kit to detect the presence of anti-pneumococcal protein and CPs antibodies, for example, anti-CPs 1, 4, 5, 6B, 9V, 14, 18C, 19F and 23F and anti-pneumococcal surface protein A antibodies, or anti-meningococcal protein and CPs antibodies, for example. anti-CPs A, B, C, Y and W-135 and anti-meningococcal OMP class 1 antibodies.
In an additional aspect of the present invention, there is provided a method of determining the presence of multivalent immunogenic conjugate molecule in a sample, comprising the steps of:
(a) immunizing a subject with an immunogenic conjugate molecule as provided herein to produce antibodies specific for the carbohydrate fragments;
(b) isolating the carbohydrate fragment specific antibodies;
(c) contacting the sample with the isolated antibodies to produce complexes comprising any said multivalent immunogenic molecules present in the sample and said isolated carbohydrate fragment specific antibodies; and
(d) determining production of the complexes.
A further aspect of the present invention provides a diagnostic kit for determining the presence of anti-bodies to a multivalent immunogenic molecule as provided herein in a sample, comprising:
(a) the multivalent immunogenic molecule;
(b) means for contacting the multivalent molecule with the sample to produce complexes comprising the multivalent molecule and any said antibodies present in the sample; and
(c) means for determining production of the complexes.
A further aspect of the present invention provides a diagnostic kit for determining the presence of a multivalent immunogenic molecule in a sample, comprising:
(a) antibodies specific for carbohydrate fragments of the multivalent immunogenic molecule;
(b) means for contacting the antibodies with the sample to produce complexes comprising multivalent immunogenic molecules and the antibodies; and
(c) means for determining the production of the complex.
The present invention also extends to the use of a mixture of anti-PRP, anti-pneumococcal CPs and anti-meningococcal CPs antibodies as a component in a diagnostic immunoassay kit to detect the presence of Hib, S. pneumoniae and N. meningitidis in biological specimens, such as serum samples.